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I.V. catheter placement is one of the most common causes of procedural pain in children. Interventions to address this pain are readily available but inconsistently used in practice. The focus of this article is to identify and encourage best practice for pain mitigation in peripheral I.V. catheter placement in children.

One of the most important functionalities of the atomically thin insulator hexagonal boron nitride (hBN) is its ability to chemically and electronically decouple functional materials from highly reactive surfaces. It is therefore of utmost importance to uncover its structural properties on surfaces on an atomic and mesoscopic length scale. In this paper, we quantify the relative coverages of structurally different domains of a hBN layer on the Ni(111) surface using low-energy electron microscopy and the normal incidence x-ray standing wave technique. We find that hBN nucleates on defect sites of the Ni(111) surface and predominantly grows in two epitaxial domains that are rotated by ${60}^{\ensuremath{\circ}}$ with respect to each other. The two domains reveal identical adsorption heights, indicating a similar chemical interaction strength with the Ni(111) surface. The different azimuthal orientations of these domains originate from different adsorption sites of N and B. We demonstrate that the majority ($\ensuremath{\approx}70%$) of hBN domains exhibit a $(\mathrm{N},\mathrm{B})=(\mathrm{top},\mathrm{fcc})$ adsorption site configuration while the minority ($\ensuremath{\approx}30%$) show a $(\mathrm{N},\mathrm{B})=(\mathrm{top},\mathrm{hcp})$ configuration. Our study hence underlines the crucial role of the atomic adsorption configuration in the mesoscopic domain structures of in situ fabricated two-dimensional materials on highly reactive surfaces.

Schuster C, Malinowsky K, Liebmann S, Berg D, Wolff C, Tran K, Schott C, Reu S, Neumann J, Faber C, Hofler H, Kirchner T, Becker K-F & Hlubek F (2012) Histopathology 60, E37–E50 Antibody validation by combining immunohistochemistry and protein extraction from formalin-fixed paraffin-embedded tissues Aims: Personalized cancer treatment strategies depend on comprehensive and detailed characterization of individual human malignancies. Clinical pathology, particularly immunohistochemical evaluation of biomarkers in tissues, is considered to be the approved standard for diagnostic and therapeutic decisions, having a direct influence on patient management and therapy. Although antibody-based approaches are established and integrated successfully into both clinical and research applications, for personalized treatment regimens new demands have been placed on the quality, reproducibility and accuracy of antibody-based assays. To ensure the accuracy of specific antigen detection in immunohistochemistry, we introduce a novel approach for antibody validation. Methods and results: In a tandem approach we used the same archival tissue of interest for antibody validation by combining extraction of immunoreactive proteins from formalin-fixed, paraffin-embedded tissue with Western blot analysis and immunohistochemistry. This procedure allows for specification of the antigen detected and for localization of the protein in the tissue. Of the 32 antibodies tested used in research and routine diagnostics, 19 showed reliable specificity in both assays. Conclusion: This study emphasizes the advantage of combining suitable methods to ensure reproducibility and specific antigen detection. Based on our results, we propose a novel step-by-step strategy to validate antibody specificity and reduce variability of immunohistochemical results.

Pancreatic endocrine tumors (PETs) may be part of hereditary multiple endocrine neoplasia type 1 (MEN1) syndrome. While MEN1 gene mutation is the only ascertained genetic anomaly described in PETs, no data exist on the cellular localization of MEN1-encoded protein, menin, in normal pancreas and PETs. A total of 169 PETs were used to assess the i) MEN1 gene mutational status in 100 clinically sporadic PETs by direct DNA sequencing, ii) immunohistochemical expression of menin in normal pancreas and 140 PETs, including 71 cases screened for gene mutations, and iii) correlation of these findings with clinical–pathological parameters. Twenty-seven PETs showed mutations that were somatic in 25 patients and revealed to be germline in 2 patients. Menin immunostaining showed strong nuclear and very faint cytoplasmic signal in normal islet cells, whereas it displayed abnormal location and expression levels in 80% of tumors. PETs harboring MEN1 truncating mutations lacked nuclear protein, and most PETs with MEN1 missense mutations showed a strong cytoplasmic positivity for menin. Menin was also misplaced in a significant number of cases lacking MEN1 mutations. In conclusion, the vast majority of PETs showed qualitative and/or quantitative alterations in menin localization. In 30% of cases, this was associated with MEN1 mutations affecting sequences involved in nuclear localization or protein–protein interaction. In cases lacking MEN1 mutations, the alteration of one of the menin interactors may have prevented its proper localization, as suggested by recent data showing that menin protein shuttles between the nucleus and cytoplasm and also affects the subcellular localization of its interactors.

Deregulated signal pathways are promising targets for cancer therapy as they can drive tumour growth, sur- vival, invasion, and metastasis. In most hospitals around the world, histopathological cancer diagnosis is based upon for- malin-fixed and paraffin-embedded (FFPE) tissues. In this study, we evaluated whether phosphorylated signaling proteins can be extracted from cells and tissues after formalin fixation. Three epidermal growth factor (EGF) receptor-positive cell lines were stimulated with EGF for various time points and then either fixed with formalin or left unfixed before protein extraction. Using Western blot, we were able to detect all phosphorylated proteins analyzed, including activated (phos- phorylated) extracellular signal-regulated kinase (ERK) 1 and 2. There were no differences between formalin-fixed and unfixed cells. Because ERK1/2 kinases are involved in HER2-mediated signal transduction, we applied our methodology to study ERK1/2 and phospho-ERK1/2 in 20 primary breast cancers using protein lysate microarray technology. We found a statistically significant correlation between HER2 status and ERK1/2 expression; however, activation (phosphory- lation) of ERK1/2 was independent of HER2 expression. Our study demonstrates the successful extraction of phosphory- lated proteins from FFPE tissue samples. Monitoring phosphorylated signaling proteins in FFPE tissues, as applied here, may allow us in the near future to infer the activity levels of proteins in particular pathways as starting point for the design of individual combination therapy regimens without changing the routine clinical workflow for tissue analysis.

Epithelial–mesenchymal transition (EMT) involving down-regulation of E-cadherin is known to play an important role in tumour progression. The aim of our study was to investigate the mRNA expression of two EMT regulators—Slug and E12/E47—in primary human gastric carcinomas and to compare this with the expression of E-cadherin and other EMT regulators (Snail, Twist, and SIP1). We studied a series of 59 gastric carcinomas by real-time quantitative RT-PCR in formalin-fixed and paraffin-embedded tissues. Thirty-four cases (58%) showed Slug up-regulation in the tumour; reduced or negative expression of E-cadherin was present in 24 of these (71%, p < 0.0001). Twenty-one cases (36%) showed E12/E47 up-regulation that was not significantly associated with E-cadherin down-regulation (p = 0.5734). Slug up-regulation accompanied by E-cadherin down-regulation correlated with the presence of distant metastases (p = 0.0029) and with advanced pTNM stages (p = 0.0424). A statistically significant association was found between Slug up-regulation and the expression of SIP1 in intestinal (p = 0.0014) and Snail in diffuse (p = 0.0067) carcinomas. We present the first study integrating the analysis of several EMT regulators in primary gastric carcinomas and conclude that Slug up-regulation is associated with E-cadherin down-regulation in diffuse and intestinal-type gastric carcinoma, and that this effect could be complemented by the presence of other EMT regulators. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Recent genomic studies revealed a high rate of recurrent mutations in the RAS pathway in primary rhabdomyosarcoma (RMS) samples. In the present study, we therefore investigated how oncogenic RAS mutants impinge on the regulation of cell death of RMS13 cells. Here, we report that ectopic expression of NRAS12V, KRAS12V, or HRAS12V protects RMS13 cells from oxidative stress-induced cell death. RMS13 cells engineered to express NRAS12V, KRAS12V, or HRAS12V were significantly less susceptible to loss of cell viability upon treatment with several oxidative stress inducers including the thioredoxin reductase inhibitor Auranofin, the glutathione (GSH) peroxidase 4 inhibitor RSL3 or Erastin, an inhibitor of the cysteine/glutamate amino acid transporter system [Formula: see text] that blocks GSH synthesis. Notably, addition of Ferrostatin-1 confers protection against Erastin- or RSL3-induced cytotoxicity, indicating that these compounds trigger ferroptosis, an iron-dependent form of programed cell death. Furthermore, RMS13 cells overexpressing oncogenic RAS mutants are significantly protected against the dual PI3K/mTOR inhibitor PI103, whereas they are similarly sensitive to DNA-damaging drugs such as Doxorubicin or Etoposide. This suggests that oncogenic RAS selectively modulates cell death pathways triggered by cytotoxic stimuli in RMS13 cells. In conclusion, our discovery of an increased resistance to oxidative stress imposed by oncogenic RAS mutants in RMS13 cells has important implications for the development of targeted therapies for RMS.

In our study, we aimed to investigate the expression of N-cadherin and E-cadherin and their dependency on epithelial-mesenchymal transition regulators SNAI1, SIP1 and TWIST in human colon cancer. Expression of E-cadherin and N-cadherin was examined by immunohistochemistry in 80 colon carcinomas by using paraffin embedded and formalin fixed tissues. Those cases were partly analyzed for mRNA expression of N-cadherin (42 cases), TWIST (18 cases), SNAI1 (25 cases) and SIP1 (25 cases) by real-time quantitative RT-PCR. Additionally, colon carcinomas that showed amplification of 20q13, the localization of the human SNAI1 gene, were examined. We found cytoplasmic and/or membrane-associated immunoreactivity of N-cadherin in 35/80 (44%) of the cases. However, there was no correlation to upregulated TWIST mRNA levels, as we have shown previously for diffuse-type gastric cancers with abnormal N-cadherin expression. Reduced and/or cytoplasmic E-cadherin immunoreactivity was detected in 19% (15/80) of the cases. Expression of SNAI1 or SIP1 mRNA was not seen in any of the 25 cases analyzed. There was no correlation between amplification of 20q13 and SNAI1 mRNA expression. Remarkably, N-cadherin was almost exclusively expressed in those cases showing normal E-cadherin immunoreactivity, suggesting a mutual exclusion between abnormal E-cadherin reduction and upregulation of N-cadherin. For the first time, we postulate a role for N-cadherin in primary colon cancer progression, which may be similar to the effect discovered by others in breast cancer cell lines, where coexpressed N-cadherin can exert a dominant function over E-cadherin's adhesive function and thus promote tumor invasiveness.

E-cadherin mutations are found in 50% of diffuse-type gastric carcinoma, but not in intestinal gastric carcinoma. Because cell-cell adhesion mediated by E-cadherin plays an important role in epithelial cell survival, E-cadherin mutations could alter the apoptotic behavior of tumor cells. p53 and Bcl-2 family members are also important regulators of cellular apoptosis. This is the first study that investigates the relationship between E-cadherin gene mutation and p53 gene mutation, p53 accumulation, Bcl-2 expression, and Ki-67 expression in diffuse-type gastric carcinoma (24 cases, E-cadherin mutation status: wild-type in 8 patients and mutant in 16 patients). The mutation status of exons 5-8 of p53 was analyzed by denaturing high pressure liquid chromatography (DHPLC) in formalin-fixed, paraffin-embedded tumor sections, followed by direct sequencing of cases with aberrant chromatographic patterns. p53 mutations were found in 1 of 8 tumors without E-cadherin mutation (12.5%) and in 1 of 16 tumors with E-cadherin mutation (6.3%), a difference that was not statistically significant (p = 1.00). p53 accumulation was found in 8 of 24 tumors (33.3%) by immunohistochemical staining. p53 accumulation was significantly more frequent in tumors without E-cadherin mutations (5 of 8 tumors, 62.5%) than in gastric carcinoma tissues with E-cadherin mutations (3 of 16 tumors, 18.8%, p = 0.03). Bcl-2 staining was not observed in gastric carcinoma cells without E-cadherin mutations, but was detectable in 5 of 16 tumors with E-cadherin mutations (31.3%), a difference that was not statistically significant (p = 0.13). No relationship was observed between Ki-67 staining and the E-cadherin mutation status (p = 1.00). These data suggest that the presence of E-cadherin mutations can significantly alter the accumulation of the apoptosis-regulating p53 protein, whereas no correlation with the p53 mutation status or with Ki-67 staining was observed.

The aim of our study was to evaluate the quality of histo- and cytomorphological features of PAXgene-fixed specimens and their suitability for histomorphological classification in comparison to standard formalin fixation. Fifteen colon cancer tissues were collected, divided into two mirrored samples and either formalin fixed (FFPE) or PAXgene fixed (PFPE) before paraffin embedding. HE- and PAS-stained sections were scanned and evaluated in a blinded, randomised ring trial by 20 pathologists from Europe and the USA using virtual microscopy. The pathologists evaluated histological grading, histological subtype, presence of adenoma, presence of lymphovascular invasion, quality of histomorphology and quality of nuclear features. Statistical analysis revealed that the reproducibility with regard to grading between both fixation methods was rather satisfactory (weighted kappa statistic (k w) = 0.73 (95 % confidence interval (CI), 0.41–0.94)), with a higher agreement between the reference evaluation and the PFPE samples (k w = 0.86 (95 % CI, 0.67–1.00)). Independent from preservation method, inter-observer reproducibility was not completely satisfactory (k w = 0.60). Histomorphological quality parameters were scored equal or better for PFPE than for FFPE samples. For example, overall quality and nuclear features, especially the detection of mitosis, were judged significantly better for PFPE cases. By contrast, significant retraction artefacts were observed more frequently in PFPE samples. In conclusion, our findings suggest that the PAXgene Tissue System leads to excellent preservation of histomorphology and nuclear features of colon cancer tissue and allows routine morphological diagnosis.

Protein phosphorylation controls the activity of signal transduction pathways regulated by kinases and phosphatases. Little is known, however, about the impact of preanalytical factors, for example, delayed times to tissue fixation, on global phosphoprotein levels in tissues. The aim of this study was to characterize the potential effects of delayed tissue preservation (cold ischemia) on the levels of phosphoproteins using targeted and nontargeted proteomic approaches. Rat and murine liver samples were exposed to different cold ischemic conditions ranging from 10 to 360 min prior to cryopreservation. The phosphoproteome was analyzed using reverse phase protein array (RPPA) technology and phosphoprotein-enriched quantitative tandem mass spectrometry (LC-MS/MS). RPPA analysis of rat liver tissues with long (up to 360 min) cold ischemia times did not reveal statistically significant alterations of specific phosphoproteins even though nonphosphorylated cytokeratin 18 (CK18) showed increased levels after 360 min of delay to freezing. Keeping the samples on ice prior to cryopreservation prevented this effect. LC-MS/MS-based quantification of 1684 phosphorylation sites in rat liver tissues showed broadening of their distribution compared to time point zero, but without reaching statistical significance for individual phosphosites. Similarly, RPPA analysis of mouse liver tissues with short (

Reverse phase protein array (RPPA) is a very suitable technique to analyze large numbers of proteins in small samples like for example tumor biopsies. Beside their small size another major hindrance for the analysis of proteins from biopsies is the extraction of proteins from formalin-fixed and paraffin-embedded (FFPE) tissues. Here we describe a protocol, allowing quantitative extraction of large numbers of proteins from FFPE tissues and their subsequent analysis by RPPA. To elucidate the role of epidermal growth factor receptor (EGFR) signalling in ovarian cancer, we analyzed 23 primary tumors and corresponding metastases for the expression of 25 proteins involved in EGFR signalling with special emphasis on epithelial-mesenchymal transition (EMT). We found a significant correlation of Snail with EGFR((Tyr1086)) and p38 MAPK((Thr180/Tyr182)) in primary ovarian carcinoma and with EGFR((Tyr1086)) in their corresponding metastases. Additionally, we showed that high expression levels of the E-cadherin repressor Snail in primary tumors combined with high expression levels of the pp38 MAPK((Thr180/Tyr182)) in metastasis lead to an increased risk for death in ovarian carcinoma patients.

Precise quantitation of protein biomarkers in clinical tissue specimens is a prerequisite for accurate and effective diagnosis, prognosis, and personalized medicine. Although progress is being made, protein analysis from formalin-fixed and paraffin-embedded tissues is still challenging. In previous reports, we showed that the novel formalin-free tissue preservation technology, the PAXgene Tissue System, allows the extraction of intact and immunoreactive proteins from PAXgene-fixed and paraffin-embedded (PFPE) tissues. In the current study, we focused on the analysis of phosphoproteins and the applicability of two-dimensional gel electrophoresis (2D-PAGE) and enzyme-linked immunosorbent assay (ELISA) to the analysis of a variety of malignant and non-malignant human tissues. Using western blot analysis, we found that phosphoproteins are quantitatively preserved in PFPE tissues, and signal intensities are comparable to that in paired, frozen tissues. Furthermore, proteins extracted from PFPE samples are suitable for 2D-PAGE and can be quantified by ELISA specific for denatured proteins. In summary, the PAXgene Tissue System reliably preserves phosphoproteins in human tissue samples, even after prolonged fixation or stabilization times, and is compatible with methods for protein analysis such as 2D-PAGE and ELISA. We conclude that the PAXgene Tissue System has the potential to serve as a versatile tissue fixative for modern pathology.

Personalized cancer treatment strategies depend on comprehensive and detailed characterization of individual human malignancies. Clinical pathology, particularly immunohistochemical evaluation of biomarkers in tissues, is considered to be the approved standard for diagnostic and therapeutic decisions, having a direct influence on patient management and therapy. Although antibody-based approaches are established and integrated successfully into both clinical and research applications, for personalized treatment regimens new demands have been placed on the quality, reproducibility and accuracy of antibody-based assays. To ensure the accuracy of specific antigen detection in immunohistochemistry, we introduce a novel approach for antibody validation. In a tandem approach we used the same archival tissue of interest for antibody validation by combining extraction of immunoreactive proteins from formalin-fixed, paraffin-embedded tissue with Western blot analysis and immunohistochemistry. This procedure allows for specification of the antigen detected and for localization of the protein in the tissue. Of the 32 antibodies tested used in research and routine diagnostics, 19 showed reliable specificity in both assays. This study emphasizes the advantage of combining suitable methods to ensure reproducibility and specific antigen detection. Based on our results, we propose a novel step-by-step strategy to validate antibody specificity and reduce variability of immunohistochemical results.

In most hospitals around the world FFPE (formalin fixed, paraffin embedded) tissues have been used for diagnosis and have subsequently been archived since decades. This has lead to a sizeable pool of this kind of tissues. Till quite recently it was not possible to use this congeries of samples for protein analysis, but now several groups described successful protein extraction from FFPE tissues. In this chapter, we describe a protein extraction protocol established in our laboratory combined with the use of reverse phase protein microarray.

In most hospitals worldwide formalin-fixation and paraffin-embedding (FFPE) is the standard tissue fixation and storage method. The analysis and extraction of full-length and immunoreactive proteins from FFPE tissues was long believed to be impossible due to protein cross-linking during fixation. Nevertheless, in recent years, several research groups have successfully established protocols to extract proteins from FFPE tissues. For use in clinical in vitro diagnostics the extraction protocol should not be too complex and has to be compatible with downstream molecular analysis such as Western-blot, reverse phase protein array, or mass-spectrometry. Here we describe a protocol for protein extraction from FFPE tissues that fulfills these requirements.

In most hospitals around the world FFPE (formalin fixed, paraffin embedded) tissues have been used for diagnosis and have subsequently been archived since decades. This has lead to a sizeable pool of this kind of tissues. Till quite recently it was not possible to use this congeries of samples for protein analysis, but now several groups described successful protein extraction from FFPE tissues. In this chapter, we describe a protein extraction protocol established in our laboratory combined with the use of reverse phase protein microarray.

Reverse-phase protein microarrays represent a very promising high-throughput technology to monitor changes in protein expression over time, before and after treatment, between disease and nondisease states, and between responders and nonresponders. This array format allows analysis of multiple samples for the expression of one protein under the same experimental conditions at the same time. Moreover, it is suited for signal transduction profiling of small numbers of cultured cells or cells isolated by laser capture microdissection from human biopsies. After protein extraction (see Chap. 37), each patient sample is arrayed in triplicates on nitrocellulose-coated slides in a miniature dilution curve. Validated antibodies are used to detect the proteins of interest. Thus, each analyte/antibody combination can be analyzed in the linear dynamic range.

Formalin fixation and paraffin embedding is the standard technique for preserving biological material for both storage and histological analysis. Although recent progress has been made in the molecular analysis of formalin-fixed, paraffin-embedded (FFPE) tissues, proteomic applications are a special challenge due to the cross-linking property of formalin. Here we present the results of a new formalin-free tissue fixative, PAXgene, and demonstrate successful extraction of nondegraded and immunoreactive protein for subsequent standard protein assays, such as Western blot analysis and reverse-phase protein arrays. High amounts of protein can be obtained from PAXgene-fixed, paraffin-embedded (PFPE) mouse liver and human spleen, breast, duodenum, and stomach tissues, similar to frozen material. By Western blot analysis, we found that the detection of membrane, cytoplasmic, nuclear, and phosphorylated protein from PAXgene-fixed human tissue samples was comparable to cryopreserved samples. Furthermore, the distribution of protein in PAXgene-fixed human tissue specimens is adequate for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry for in situ proteomic analysis. Taken together, we demonstrate here that PAXgene has great potential to serve as a novel multimodal fixative for modern pathology, enabling extensive protein biomarker studies on clinical tissue samples.

Formalin fixed and paraffin embedded (FFPE) tissues are the basis for histopathological diagnosis of many diseases around the world. For translational research and routine diagnostics, protein analysis from FFPE tissues is very important. We evaluated the potential influence of six histological stains, including hematoxylin (Mayer and Gill), fast red, light green, methyl blue and toluidine blue, for yield, electrophoretic mobility in 1-D gels, and immunoreactivity of proteins isolated from formalin-fixed breast cancer tissues. Proteins extracted from stained FFPE tissues using a recently established technique were compared with proteins obtained from the same tissues but without prior histological staining. Western blot and quantitative protein lysate microarray analysis demonstrated that histological staining can result in decreased protein yield but may not have much influence on immunoreactivity and electrophoretic mobility. Interestingly, not all staining protocols tested are compatible with subsequent protein analysis. The commonly used hematoxylin staining was found to be suitable for multiplexed quantitative protein measurement technologies although protein extraction was less efficient. For best results we suggest a guided protein extraction method, in which an adjacent hematoxylin/eosin-stained tissue section is used to control dissection of an unstained specimen for subsequent protein extraction and quantification for research and diagnosis.

Owing to its cross-linking effects, it is currently believed that formalin fixation of routinely processed tissues in the clinic prevents protein extraction and profiling. The aim of our study was to develop a robust, fast, standardized, and easy to use technique for the solubilization of non-degraded, full length, and immunoreactive proteins from formalin-fixed tissues for western blot and protein microarray analysis. Sections of routinely processed formalin-fixed and paraffin-embedded tissues of various origin were analysed. After deparaffination, tissues were manually dissected from the slides and transferred into an optimized protein extraction buffer system. Proteins were solubilized and subsequently analysed by western blot and reverse phase protein microarrays. We succeeded in isolating non-degraded, soluble, and immunoreactive proteins from routinely processed formalin-fixed tissues. We were able to detect membrane, cytoplasmic and nuclear proteins at the expected molecular weight. No differences were found in the protein yield and protein abundances between fresh frozen and formalin-fixed tissues. Using western blots and reverse phase protein microarrays, the receptor tyrosine kinase HER2, an important protein target for antibody based cancer treatment, was reliably measured in formalin-fixed breast cancer biopsy samples when compared with measurement by immunohistochemistry and fluorescence in situ hybridization; remarkably, immunohistochemically equivocal cases (score 2+) can be categorized according to HER2 protein abundance. Our new clinically orientated multiplexed protein measurement system may be generally applicable to determine the relative abundances of known disease-related proteins in small amounts of routinely processed formalin-fixed tissue samples for research and diagnosis. This technique may also be used to identify, characterize, and validate known and new protein markers in a variety of human diseases.

In our study, we aimed to investigate the expression of N-cadherin and E-cadherin and their dependency on epithelial-mesenchymal transition regulators SNAI1, SIP1 and TWIST in human colon cancer. Expression of E-cadherin and N-cadherin was examined by immunohistochemistry in 80 colon carcinomas by using paraffin embedded and formalin fixed tissues. Those cases were partly analyzed for mRNA expression of N-cadherin (42 cases), TWIST (18 cases), SNAI1 (25 cases) and SIP1 (25 cases) by real-time quantitative RT-PCR. Additionally, colon carcinomas that showed amplification of 20q13, the localization of the human SNAI1 gene, were examined. We found cytoplasmic and/or membrane-associated immunoreactivity of N-cadherin in 35/80 (44%) of the cases. However, there was no correlation to upregulated TWIST mRNA levels, as we have shown previously for diffuse-type gastric cancers with abnormal N-cadherin expression. Reduced and/or cytoplasmic E-cadherin immunoreactivity was detected in 19% (15/80) of the cases. Expression of SNAI1 or SIP1 mRNA was not seen in any of the 25 cases analyzed. There was no correlation between amplification of 20q13 and SNAI1 mRNA expression. Remarkably, N-cadherin was almost exclusively expressed in those cases showing normal E-cadherin immunoreactivity, suggesting a mutual exclusion between abnormal E-cadherin reduction and upregulation of N-cadherin. For the first time, we postulate a role for N-cadherin in primary colon cancer progression, which may be similar to the effect discovered by others in breast cancer cell lines, where coexpressed N-cadherin can exert a dominant function over E-cadherin's adhesive function and thus promote tumor invasiveness.

E-cadherin mutations are found in 50% of diffuse-type gastric carcinoma, but not in intestinal gastric carcinoma. Because cell-cell adhesion mediated by E-cadherin plays an important role in epithelial cell survival, E-cadherin mutations could alter the apoptotic behavior of tumor cells. p53 and Bcl-2 family members are also important regulators of cellular apoptosis. This is the first study that investigates the relationship between E-cadherin gene mutation and p53 gene mutation, p53 accumulation, Bcl-2 expression, and Ki-67 expression in diffuse-type gastric carcinoma (24 cases, E-cadherin mutation status: wild-type in 8 patients and mutant in 16 patients). The mutation status of exons 5-8 of p53 was analyzed by denaturing high pressure liquid chromatography (DHPLC) in formalin-fixed, paraffin-embedded tumor sections, followed by direct sequencing of cases with aberrant chromatographic patterns. p53 mutations were found in 1 of 8 tumors without E-cadherin mutation (12.5%) and in 1 of 16 tumors with E-cadherin mutation (6.3%), a difference that was not statistically significant (p = 1.00). p53 accumulation was found in 8 of 24 tumors (33.3%) by immunohistochemical staining. p53 accumulation was significantly more frequent in tumors without E-cadherin mutations (5 of 8 tumors, 62.5%) than in gastric carcinoma tissues with E-cadherin mutations (3 of 16 tumors, 18.8%, p = 0.03). Bcl-2 staining was not observed in gastric carcinoma cells without E-cadherin mutations, but was detectable in 5 of 16 tumors with E-cadherin mutations (31.3%), a difference that was not statistically significant (p = 0.13). No relationship was observed between Ki-67 staining and the E-cadherin mutation status (p = 1.00). These data suggest that the presence of E-cadherin mutations can significantly alter the accumulation of the apoptosis-regulating p53 protein, whereas no correlation with the p53 mutation status or with Ki-67 staining was observed.

A major function of the cell-to-cell adhesion molecule E-cadherin is the maintenance of cell adhesion and tissue integrity. E-cadherin deficiency in tumours leads to changes in cell morphology and motility, so that E-cadherin is considered to be a suppressor of invasion. In this study we investigated the functional consequences of three tumour-associated gene mutations that affect the extracellular portion of E-cadherin: in-frame deletions of exons 8 or 9 and a point mutation in exon 8, as they were found in human gastric carcinomas. Human MDA-MB-435S breast carcinoma cells and mouse L fibroblasts were stably transfected with the wild-type and mutant cDNAs, and the resulting changes in localization of E-cadherin, cell morphology, strength of calcium-dependent aggregation as well as cell motility and actin cytoskeleton organization were studied. We found that cells transfected with wild-type E-cadherin showed an epitheloid morphology, while all cell lines expressing mutant E-cadherin exhibited more irregular cell shapes. Cells expressing E-cadherin mutated in exon 8 showed the most scattered appearance, whereas cells with deletion of exon 9 had an intermediate state. Mutant E-cadherins were localized to the lateral regions of cell-to-cell contact sites. Additionally, both exon 8-mutated E-cadherins showed apical and perinuclear localization, and actin filaments were drastically reduced. MDA-MB-435S cells with initial calcium-dependent cell aggregation exhibited decreased aggregation and, remarkably, increased cell motility, when mutant E-cadherin was expressed. Therefore, we conclude that these E-cadherin mutations may not simply affect cell adhesion but may act in a trans-dominant-active manner, i.e. lead to increased cell motility. Our study suggests that E-cadherin mutations affecting exons 8 or 9 are the cause of multiple morphological and functional disorders and could induce the scattered morphology and the invasive behaviour of diffuse type-gastric carcinomas.

The quality of human tissue specimens can have a significant impact on analytical data sets for biomarker research. The aim of this study was to characterize fluctuations of protein and phosphoprotein levels in human tissue samples during the preanalytical phase. Eleven intestine and 17 liver specimens were surgically resected, aliquoted, and either snap-frozen or fixed in formalin immediately or exposed to different ischemic conditions before preservation. Protein levels in the resultant samples were investigated by reverse phase protein array, Western blot analysis, and liquid chromatography tandem mass spectrometry. Our data revealed that the degree of sensitivity of proteins and phosphoproteins to delayed preservation varied between different patients and tissue types. For example, up-regulation of phospho-p42/44 MAPK in intestine samples was seen in some patients but not in others. General trends toward up- or down regulation of most proteins were not evident due to pronounced interpatient variability but signal intensities of only a few proteins, such as cytokeratin 18, were altered from baseline in postresection samples. In contrast, glyceraldehyde 3:phosphate dehydrogenase was found to be stable during periods of cold ischemia. Our study represents a proper approach for studying potential protein fluctuations in tissue specimens for future biomarker development programs.

We report four DNA variants in the gene coding for the cell adhesion molecule E-cadherin. The polymorphisms affect codons 115, 133, 582 and the 3'-non-coding region.

The cell adhesion molecule E-cadherin (CDH1; MIM# 192090) has been implicated in numerous cellular functions, ranging from controlling morphogenesis to suppressing tumor invasion. We describe 11 previously unreported somatic E-cadherin mutations in two subgroups of gastric and breast cancer showing markedly reduced homophilic cell-to-cell interactions. Using reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing of the entire coding region 5 mutations were detected in diffuse-type gastric cancer specimens. The sequence alterations include 3 missense mutations affecting exons 3, 10, and 12. Furthermore, two in-frame deletions were identified removing 63 and 9 base pairs from exon 4 and 5, respectively. In invasive Lobular breast cancer 6 E-cadherin mutations were detected after RT-PCR amplification and direct sequencing or using single strand conformation polymorphism (SSCP) analysis followed by sequencing. In addition to two nonsense mutations affecting exon 2, four out-of-frame deletions removing 115 base pairs (entire exon 2), 224 base pairs (entire exon 3), 8 base pairs from exon 12 or 1 base pair from exon 13 were seen. Our report confirms the general principle that in diffuse-type gastric cancer E-cadherin mutations result in structurally altered proteins with possible reduced adhesive functions whereas in invasive lobular breast carcinomas complete loss-of-function mutations are characteristic.

Background The availability of well-annotated prostate tissue samples through biobanks is key for research. Whereas fresh-frozen tissue is well suited for a broad spectrum of molecular analyses, its storage and handling is complex and cost-intensive. Formalin-fixed paraffin-embedded specimens (FFPE) are easy to handle and economic to store, but their applicability for molecular methods is restricted. The recently introduced Hepes-glutamic acid-buffer mediated Organic solvent Protection Effect (HOPE) is a promising alternative, which might have the potential to unite the benefits of FFPE and fresh-frozen specimen. Aim of the study was to compare HOPE-fixed, FFPE and fresh-frozen bio-specimens for their accessibility for diagnostic and research purposes. Methods 10 prostate cancer samples were each preserved with HOPE, formalin, and liquid nitrogen and studied with in-situ and molecular methods. Samples were H&E stained, and assessed by immunohistochemistry (i.e. PSA, GOLPH2, p63) and FISH (i.e. ERG rearrangement). We assessed DNA integrity by PCR, using control genes ranging from 100 to 600 bp amplicon size. RNA integrity was assessed through qRT-PCR on three housekeeping genes (TBP, GAPDH, β-actin). Protein expression was analysed by performing western blot analysis using GOLPH2 and PSA antibodies. Results Of the HOPE samples, morphologic quality of H&E sections, immunohistochemical staining, and the FISH assay was at least equal to FFPE tissue, and significantly better than the fresh-frozen specimens. DNA, RNA, and protein analysis of HOPE samples provided similar results as compared to fresh-frozen specimens. As expected, FFPE-samples were inferior for most of the molecular analyses. Conclusions This is the first study, comparatively assessing the suitability of these fixation methods for diagnostic and research utilization. Overall, HOPE-fixed bio-specimens combine the benefits of FFPE- and fresh-frozen samples. Results of this study have the potential to expand on contemporary prostate tissue biobanking approaches and can serve as a model for other organs and tumors.